Elucidation of the Mechanism of Vitamin D Degradation in Powdered Milk

Abstract

Vitamin D is an essential nutrient which plays a significant role in many biochemical functions such as bone renewal, cell growth and blood cell formation. Vitamin D deficiency, which is now a global public-health problem, may increase the risk of many diseases. This nutrient can be synthesized in the body through exposure to sunlight, however, several factors such as seasonal changes, living at high latitude and aging can impair this process. Since only a limited number of foods contain vitamin D, the fortification of food with this nutrient, mostly as vitamin D3, has been in the centre of attention. Among different food sources, a fatty vehicle like whole milk is a good target for fat-soluble vitamins fortification such as vitamin D. In a successful fortification program, the stability of micronutrients added to the food is important and may depends on several factors. In terms of vitamin D, significant disagreements regarding its stability to light, oxidation and acidity have been stated. Previous studies also reported the inconsistencies between analysed values and claimed amounts of vitamin D in fortified products. The paucity of literature on the stability of vitamin D3 suggests that more studies are needed to understand the fate of this nutrient especially in complex matrices like food systems. This project aimed to elucidate the mechanism of vitamin D3 degradation in fortified milk powders. The effects of different physicochemical stresses upon vitamin D3 were studied with the aim of creating markers of degradation pathways in situ. The work included generating isomers of degradation products of vitamin D3 and the development of analytical methods to identify such products. Further, long-term storage trials were conducted to monitor vitamin D3 stability in simulated whole milk powder with simple matrices. The influence of processing conditions (heat treatment) and storage conditions (time and temperature) on lipid oxidation and vitamin D3 degradation in simulated whole milk powders (SWMPs) were investigated. Vitamin D3 degradation during storage of whole milk powder could be attributed to the oxidation of vitamin D3 mediated by lipid oxidation. Vitamin D, which has similar structures to those of olefin compounds, could be caught up in the lipoxidation reactions of unsaturated fats. Finally, vitamin D3 oxidation products (VDOPs) were identified and quantified in stored SWMPs. An optimized method for extraction of VDOPs in SWMPs was based on a liquid-liquid extraction followed by a solid phase extraction to separate these compounds from vitamin D3 and lipid matrices. VDOPs with one additional oxygen, 1-hydroxy- vitamin D3 and 7,8-epoxy-vitamin D3, and two additional oxygen atoms, 1-keto-7,8-epoxy- vitamin D3 and 1-hydroxy-7,8-epoxyvitamin D3, were identified using LC/MS and LC/MSn techniques. Methods based on high mass accuracy MS2 and MSn analysis have been developed to identify their fragmentation rules by incorporating MS data with in silico calculated MS fragmentation pathways. Finally, PTADderivatized VDOPs were quantified using UHPC-QQQ-MS/MS in MRM mode.